Engraftment has been established in nonsensitized major histocompatibility complex (MHC)-matched recipients with nonmyeloablative conditioning regimens. Recipients previously transfused with blood products become sensitized to donor minor histocompatibility antigens, increasing the risk of graft rejection. Patients with inherited red blood cell diseases require blood transfusions and have a higher probability of graft rejection. In this proposal, nonmyeloablative conditioning regimens will be developed for sensitized recipients which 1) lack the toxicities characteristic of myeloablative regimens, and 2) could be safely administered in an outpatient setting. The development of the nonmyeloablative regimen for sensitized patients will be based on two hypotheses: 1) host-versus-graft (HVG) reactions of sensitized recipients can be suppressed with immunosuppressive agents other than high-dose chemoradiotherapy; 2) T cells from the graft can suppress the host immune system including sensitized immune effector cells. These hypotheses will be tested and nonmyeloablative regimens will be developed in a preclinical canine model of transfusion-induced sensitization. Sensitizing recipients with blood transfusions prior to transplant results in uniform graft rejection with conventional high-dose conditioning. In Aim 1, it will be determined if further stepwise intensification of pretransplant immunosuppression in addition to CSP will successfully promote engraftment in a dose de-escalation study of TBI. Posttransplant immunosuppression (MMF/CSP) will be assessed separately to determine if it prevents graft rejection in sensitized recipients at TBI 920 cGy. If effective, intensification of posttransplant immunosuppression will be done at the maximal TBI dose at which graft rejection occurs at the completion of Aim 1A. In Aim 2, it will be determined if promoting GVH will achieve engraftment by suppressing sensitized host T cells and "creating space" in the marrow. If these studies are successful, then donor T cells will be ex vivo expanded and transduced with a "suicide (HSVtk) gene" for prevention of severe GVHD. In Aim 3, the optimal immunosuppressive regimen will be combined with a GVH enhancing regimen. The ultimate goal of this proposal is the elimination of cytotoxic agents from the conditioning regimen. By lessening the morbidity and mortality associated with conventional conditioning, these studies could significantly change the management of selected inherited red blood cell diseases.